I. Field of the Invention
This invention relates to selectin variants. The invention further relates to nucleic acids encoding, and to methods and means for preparing these variants.
II. Description of Background and Related Art
The selectins are cell adhesion molecules that are unified structurally by the inclusion of lectin, egf-like and complement binding-like domains [Bevilacqua, M. P., et al., Science 243, 1160-1165 (1989); Johnson, et al., Cell 26, 1033-144 (1989); Lasky, L. A., Cell 56, 1045-1055 (1989); Siegelman, M. et al., Science 243, 1165-1172 (1989); Stoolman, L. M., Cell 56; 907-910 (1989)], and functionally by their ability to mediate cell binding through interactions between their lectin domains and cell surface carbohydrate ligands [Brandley, B., et al. Cell 63, 861-863 (1990); Springer, T., and Lasky, L. A. Nature 349 196-197 (1991)].
There are three members identified so far in the selectin family of cell adhesion molecules: L-Selectin (a.k.a. peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1, gp.sup.90.spsp.MEL, gp.sup.100.spsp.MEL, gp.sup.110.spsp.MEL, MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen), E-Selectin (LEC-CAM-2, LECAM-2, ELAM-1) and P-Selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM). The structures of the selectin family members are illustrated in FIG. 9.
L-Selectin is found on leukocytes and is involved with the trafficking of lymphocytes to peripheral lymphoid tissues [Gallatin et al., Nature 303, 30-34 (1983)] and with acute neutrophil-mediated inflammatory responses [Watson, S. R., Nature 349 164-167 (1991)]. The amino acid sequence of L-Selectin and the encoding nucleic acid sequence are, for example, disclosed in U.S. Pat. No. 5,098,833 issued 24 Mar. 1992. L-Selectin appears to recognize sialylated, fucosylated, sulfated carbohydrate ligand(s) on at least two endothelial glycoproteins [True, D. D., et al., J. Cell Biol. 111, 2757-2764 (1990); Imai, Y. et al., J. Cell Biol. 113 1213-1221 (1991)], one of which has recently been cloned [Lasky, L. A. et al., Cell (1992), in press and copending U.S. application Ser. No. 07/834,902 filed 13 Feb. 1992].
E-Selectin is an endothelial adhesion molecule that is induced by various inflammatory stimuli [Bevilacqua, P. P. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 9238-9242 (1987); Luscinskas, F. W. et al., J. Immunol. 142 2257-2263 (1989); Kuijpers, T. W. et al., J. Immunol. 147 1369-1376 (1991)]. A cloned gene encoding E-Selectin (ELAM-1) is disclosed in U.S. Pat. No. 5,081,034 issued 14 Jan. 1992. E-Selectin recognizes the neutrophil and monocyte cell surface carbohydrate, sialyl Lewis x (sLex) [Lowe J. B. et al., Cell 63 475-484 (1990); Phillips, M. L. et al., Science 250 1130-1132 (1990); Walz, G. A. et al. Science 250 1132-1135 (1990); Tiemeyer, M. et al., Proc. Natl. Acad. Sci. U.S.A. 88 1138-1142 (1991)], and in addition, may also be involved with the recognition of an sLex-like carbohydrate on the surface of a skin-homing subset of lymphocytes [Picker, L. J. et al., Nature 349 796-799 (1991); Shimizu, Y. et al., Nature 349, 799-802 (1991)]. The minimum sized sLex-related carbohydrate recognized by E-Selectin is a tetrasaccharide of the structure Sialic Acid alpha2-3 Galactose beta 1-4 N-Acetyl Glucosamine (Fucose alpha 1-3) [Tyrrell, D. et al., Proc. Natl. Acad, Sci. U.S.A. 88, 10372-10376 (1991)].
P-Selectin is found in alpha granules of platelets and Weible-Palade bodies of endothelial cells [Bonfanti, R. et al., Blood 73, 1109-1112 (1989); McEver, R. et al., J. Clin. Inv. 84, 92-99 (1989)]. Its surface expression is induced within minutes of exposure to thrombin, substance P, histamine or peroxide, and it appears to recognize a carbohydrate that is either identical to or closely related to sLex on both neutrophil and monocyte cell surfaces [Larsen, E. et al., Cell 59, 305-312 (1989); Larsen, E. et al. Cell 63, 467-474 (1990); Moore, K. L. et al. J. Cell Biol. 112, 491-499 (1991); Polley, M. J. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 6224-6228 (1991)]. The P-Selectin amino acid and the encoding nucleotide sequences are disclosed by Johnston et al., Cell 56, 1033-1044 (1989).
The lectin domains of L-, E- and P-Selectins show remarkable sequence homology and structural similarity. Particularly noteworthy is the conservation of cysteine (Cys) residues at amino acid positions 19, 90, 109 and 117 of the selectin lectin domains, which results in a three dimensional structure comprising two-disulfide bonded loops defined by disulfide bonds formed between Cys19 and Cys117, and Cys90 and Cys109, respectively.
Much evidence has accumulated to indicate similarities in the nature of the carbohydrate ligands seen by selectins. In the case of all three selectins, the adhesive interactions between their lectin domains and carbohydrate ligands require the presence of alpha 2-3 linked sialic acid fucose residues [Brandley, B. et al., Cell 63, 861-863 (1990); Corrall, L. et al., Biochem. Biphys. Res. Commun. 172, 1349-1352 (1990); Springer, T. and Lasky, L. A. Nature 349 196-197 (1991); Tyrrell, D. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 10372-10376 (1991)]. The adhesive interactions between selectin lectin domains and their carbohydrate ligands may be relatively weak, since L- and P-Selectin have been shown to be involved in the relatively low affinity "rolling" of leukocytes along the endothelium during inflammatory responses [Lawrence, M. B. et al., Cell 65, 859-873 (1991); Ley, K. et al. Blood 77 (12), 2553-2555 (1991); Von Andrian, V. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 1538-1542 (1991)].
The molecular details of the interactions between selectin lectin domains and their carbohydrate ligands are poorly understood. The fact that all three selectins require sialic acid for adhesion, when coupled with the finding that certain negatively charged carbohydrate polymers, such as fucoidin, dextran sulfate, and polyphosphomannan ester, are effective inhibitors of some selectin-mediated cell adhesion, is consistent with the involvement of positively-charged amino acids in carbohydrate recognition. However, that such protein-sialic acid interactions can also be Accomplished by non-charged side chains is suggested by crystallographic analysis of the low-affinity interaction between the influenza hemagglutinin glycoprotein, which is not related to type C lectins, and its cell surface ligand, sialic acid, which has revealed that this interaction involves a diversity of amino acid side chains, none of which are positively charged [Weis, W. et al., Science 254, 1608-1615 (1985)]. That a simple face or pocket of the E-, and potentially P- [Polley, M. J. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 6224-6228 (1991)] Selectin lectin domain is involved with the recognition of sLex is suggested by NMR solution analyses of the sLex structure which demonstrate that the critical sialic acid and fucose residues both point to one face of this carbohydrate ligand and are separated by .about.10 angstroms, while an inactive form of this carbohydrate (with a 2-6 linked sialic acid) has these two important functional components pointing in very different directions. A similar structural analysis of another ligand for E-Selectin, sialyl Lewis a (sLea: Sialic Acid alpha 2-3 Galactose beta 1-3 N-Acetyl Glucosamine (Fucose alpha 1-4)), has revealed that the critical sialic acid and fucose residues again point to one face of the tetrasaccharide and are separated by approximately the same distance as they are in sLex. See Berg, E. L. et al. J. Biol. Chem. 265, 14869-72 (1991); Tyrrell, D. et al. (1991), Supra.
An object of the present invention is to identify the region(s) within the amino acid sequence of selectin lectin domains that is/are critical for the interaction of selectin receptors and their ligands.
It is another object to enable the preparation of amino acid sequence variants of selectins having improved ligand binding properties, in particular increased affinity for their respective ligands as compared to the corresponding native selectin receptors.
A further object is to identify selectin lectin domain sequences that are not critical for the interaction of selectins and their respective ligands.
It is another object to provide selectin amino acid sequence variants with improved pharmacological characteristics, e.g. increased physical and/or chemical stability, increased half-life, decreased rate of in vivo clearance, decreased severity or occurrence of side effects during therapeutic use, etc., having retained or increased ligand binding affinity as compared to the corresponding native selectin.
Selectin variants with enhanced ligand binding properties have great therapeutic potential as effective inhibitors of pathologic inflammatory responses mediated by selectins by blocking the selectin-selectin ligand interaction.
These and further objects of the present invention will be apparent for one skilled in the art.